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Published January 15, 1997 | Published
Journal Article Open

Kinetic and thermodynamic consequences of the removal of the Cys-77–Cys-123 disulphide bond for the folding of TEM-1 β-lactamase

Abstract

Class A β-lactamases of the TEM family contain a single disulphide bond which connects cysteine residues 77 and 123. To clarify the possible role of the disulphide bond in the stability and folding kinetics of the TEM-1 β-lactamase, this bond was removed by introducing a Cys-77 Ser mutation, and the enzymically active mutant protein was studied by reversible guanidine hydrochloride-induced denaturation. The unfolding and refolding rates were monitored using tryptophan fluorescence. At low guanidine hydrochloride concentrations, the refolding of the wild-type and mutant enzymes followed biphasic time courses. The characteristics of the two phases were not significantly affected by the mutation. Double-jump experiments, in which the protein was unfolded in a high concentration of guanidine hydrochloride for a short time period and then refolded by diluting out the denaturant, indicated that, for both the wild-type and mutant enzymes, the two refolding phases could be ascribed to proline isomerization reactions. Equilibrium unfolding experiments monitored by fluorescence spectroscopy and far-UV CD indicated a three-state mechanism (N H U). Both the folded mutant protein (N) and, to a lesser extent, the thermodynamically stable intermediate, H, were destabilized relative to the fully unfolded state, U. Removal of the disulphide bond resulted in a decrease of 14.2 kJ/mol (3.4 kcal/mol) in the global free energy of stabilization. Similarly, the mutation also induced a drastic increase in the rate of thermal inactivation.

Additional Information

© 1997 The Biochemical Society, London. Received 17 April 1996/4 September 1996; Accepted 18 September 1996. This work was supported, in part, by the Belgian Government in the frame of a Po#le d'Attraction Interuniversitaire (PAI no. 19), an Action Concerte!e with the Belgian Government (convention 89/94-130) and the Fonds de la Recherche Scienti®que Medicale (contract no. 3.4537.88). M. V. is a fellow of the Fond pour la Formation a' la Recherche dans l 'Industrie et l'Agriculture (F.R.I.A., Brussels, Belgium).

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